Project Summary The laboratory led by PI Galia Debelouchina has the following objectives: 1) The development of structural biology methodology to study complex and dynamic biological assemblies in vitro, and 2) The extension of these methodologies for structural biology investigations in the cellular environment. Our methodology development combines solid-state nuclear magnetic resonance (NMR) spectroscopy with state-of-the-art chemical biology tools for the comprehensive description of biological systems both in vitro and in cells. We use these technologies to investigate chromatin, a complex protein-DNA polymer responsible for the packaging of genetic information in the nucleus. Over the next five years, we plan to focus on the following projects: 1) The influence of protein post-translational modifications on chromatin structure and dynamics. Modifications such as methylation and acetylation have profound effects on the compaction of the chromatin polymer and the accessibility of the packaged DNA. We plan to investigate the molecular mechanisms of chromatin compaction and decompaction imparted by these modifications at atomic resolution using solid-state NMR spectroscopy. We expect that this study will provide unique insights into the biophysical properties of the chromatin polymer and how they can be translated into functional biological outputs. 2) The molecular basis of heterochromatin formation. Heterochromatin compartments are associated with gene silencing and repetitive DNA sequences, and their formation is a vital step in cell differentiation and development. Recent hypotheses indicate that they are formed through liquid-liquid phase separation. We plan to investigate the interactions that promote phase separation and to provide the first molecular description of this process. 3) Development of NMR-based tools for structural biology in cells. Here, we plan to focus on the design and implementation of a strategy to target a specific protein in the cellular milieu and to increase its NMR signals selectively over the cellular background. Ultimately, we aim to develop a selective and sensitive NMR methodology that will allow us to record relevant structural data of endogenous amounts of cellular proteins. Such tools will provide the unprecedented opportunity to build an atomic resolution picture of the cellular milieu and to investigate changes in protein structure and dynamics as we stress, age or treat cells with therapeutic agents.